Therapeutic agents

ABSTRACT

An immunoresponsive cell, such as a T-cell expressing
         (i) a second generation chimeric antigen receptor comprising:
           (a) a signalling region;   (b) a co-stimulatory signalling region;   (c) a transmembrane domain; and   (d) a binding element that specifically interacts with a first epitope on a target antigen; and   
           (ii) a chimeric costimulatory receptor comprising
           (e) a co-stimulatory signalling region which is different to that of (b);   (f) a transmembrane domain; and   g) a binding element that specifically interacts with a second epitope on a target antigen.   
               

     This arrangement is referred to as parallel chimeric activating receptors (pCAR). Cells of this type are useful in therapy, and kits and methods for using them as well as methods for preparing them are described and claimed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/749,016, filed Jan. 30, 2018, which is a 371 of InternationalPatent Application No. PCT/GB2016/052324, filed Jul. 28, 2016, whichclaims the benefit of GB Application No. 1513540.3, filed Jul. 31, 2015,the disclosure of each is hereby incorporated by reference in itsentirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted inASCII format via EFS-Web and is hereby incorporated by reference in itsentirety. The ASCII copy, created on Jun. 11, 2016, is namedSequence_Listing.txt, and is 8000 bytes in size.

FIELD OF THE INVENTION

The present invention relates to nucleic acids encoding novel chimericantigen receptors (CARs), as well as to the CARs themselves, cellsincorporating the nucleic acids and their use in therapy, in particularto methods in which they are used to facilitate a T-cell response to aselected target.

BACKGROUND OF THE INVENTION

Chimeric antigen receptors (CARs), which may also be referred to asartificial T cell receptors, chimeric T cell receptors (TCR) or chimericimmunoreceptors are engineered receptors, are well known in the art.They are used primarily to transform immune effector cells, inparticular T-cells, so as to provide those cells with a particularspecificity. They are particularly under investigation in the field ofcancer immunotherapy where they may be used in techniques such asadoptive cell transfer. In these therapies, T-cells are removed from apatient and modified so that they express receptors specific to theantigens found in a particular form of cancer. The T cells, which canthen recognize and kill the cancer cells, are reintroduced into thepatient.

First generation CARs provide a TCR-like signal, most commonly using CD3zeta (z) and thereby elicit tumoricidal functions. However, theengagement of CD3z-chain fusion receptors may not suffice to elicitsubstantial IL-2 secretion and/or proliferation in the absence of aconcomitant co-stimulatory signal. In physiological T-cell responses,optimal lymphocyte activation requires the engagement of one or moreco-stimulatory receptors (signal 2) such as CD28 or 4-1BB. Consequently,T cells have also been engineered so that they receive a co-stimulatorysignal in a tumour antigen-dependent manner.

An important development in this regard has been the successful designof ‘second generation CARs’ that transduce a functionalantigen-dependent co-stimulatory signal in human primary T cells,permitting T-cell proliferation in addition to tumouricidal activity.Second generation CARs most commonly provide co-stimulation usingmodules derived from CD28 or 4-1BB. The combined delivery ofco-stimulation plus a CD3 zeta signal renders second generation CARsclearly superior in terms of function, when compared to their firstgeneration counterparts (CD3z signal alone). An example of a secondgeneration CAR is found in U.S. Pat. No. 7,446,190.

More recently, so-called ‘third generation CARs’ have been prepared.These combine multiple signalling domains, such as CD28+4-1BB+CD3z orCD28+OX40+CD3z, to further augment potency. In the 3rd generation CARs,the signalling domains are aligned in series in the CAR endodomain andplaced upstream of CD3z.

In general however, the results achieved with these third generationCARs have disappointingly represented only a marginal improvement over2nd generation configurations.

The use of cells transformed with multiple constructs has also beensuggested. For example, Kloss et al. Nature Biotechnology 2012,doi:10.10.38nbt.2459 describes the transduction of T-cells with a CARcomprising a signal activation region (CD3 zeta chain) that targets afirst antigen and a chimeric co-stimulatory receptor (CCR) comprisingboth CD28 and 4-1BB costimulatory regions which targets a secondantigen. The two constructs bind to their respective antigens withdifferent binding affinities and this leads to a ‘tumour sensing’ effectthat may enhance the specificity of the therapy with a view to reducingside effects.

It is desirable to develop systems whereby T-cells can be maintained ina state that they can grow, produce cytokines and deliver a kill signalthrough several repeated rounds of stimulation by antigen-expressingtumour target cells. Provision of sub-optimal co-stimulation causesT-cells to lose these effector functions rapidly upon re-stimulation,entering a state known as “anergy”. When CAR T-cells are sequentiallyre-stimulated in vitro, they progressively lose effector properties(e.g. IL-2 production, ability to proliferate) and differentiate tobecome more effector-like-in other words, less likely to manifest theeffects of co-stimulation. This is undesirable for a cancerimmunotherapy since more differentiated cells tend to have lesslongevity and reduced ability to undergo further growth/activation whenthey are stimulated repeatedly in the tumour microenvironment.

SUMMARY OF THE INVENTION

The applicants have found that effective T-cell responses may begenerated using a combination of constructs in which multipleco-stimulatory regions are arranged in distinct constructs.

According to a first aspect of the present invention, there is providedan immuno-responsive cell expressing

-   -   (i) a second generation chimeric antigen receptor comprising:        -   (a) a signalling region;        -   (b) a co-stimulatory signalling region;        -   (c) a transmembrane domain; and        -   (d) a binding element that specifically interacts with a            first epitope on a target antigen; and    -   (ii) a chimeric costimulatory receptor comprising        -   (e) a co-stimulatory signalling region which is different to            that of (b);        -   (f) a transmembrane domain; and        -   (g) a binding element that specifically interacts with a            second epitope on a target antigen.

The applicants have found that the efficacy of this system is good andin particular may be better than that achieved using conventional thirdgeneration CARs having similar elements. Constructs of the type of theinvention may be called ‘parallel chimeric activating receptors’ or‘pCAR’.

In addition, the proliferation of the cells, their ability to maintaintheir cytotoxic potency and to release IL-2 is maintained over manyrepeated rounds of stimulation with antigen-expressing tumour cells.

Without being bound by theory, the arrangement of the elements in thepCARs may be facilitating activity. For example, by definition, one ofthe co-stimulatory modules in a 3rd generation CAR must be placed awayfrom its natural location close to the inner leaflet of the plasmamembrane. This may cause it not to signal normally owing to impairedaccess to obligate membrane-associated partner molecules. Alternatively,close proximity of 2 co-stimulatory signalling modules in a 3rdgeneration CAR might lead to steric issues, preventing full engagementof one or more downstream signalling pathways. Both of these issues areavoided in the arrangement of the invention. Both the signallingmoieties (b) and (e) may be fused directly to a transmembrane domain,ensuring that they are both adjacent to the plasma membrane within thecell. Furthermore, they may be spaced: at distinct sites within the cellso that will not interact sterically with each other.

Suitable immuno-responsive cells for use in the first aspect of theinvention include T-cells such as cytotoxic T-cells, helper T-cells orregulatory T-cells and Natural Killer (NK) cells. In particular, theimmuno-responsive cell is a T-cell.

Suitable elements (a) above may include any suitable signalling region,including any region comprising anImmune-receptor-Tyrosine-based-Activation-Motif (ITAM), as reviewed forexample by Love et al. Cold Spring Harbor Perspect. Biol 2010 2(6)|a002485. In a particular embodiment, the signalling region comprises theintracellular domain of human CD3 [zeta] chain as described for examplein U.S. Pat. No. 7,446,190, or a variant thereof.

In particular, this comprises the domain, which spans amino acidresidues 52-163 of the full-length human CD3 zeta chain. It has a numberof polymorphic forms (e.g. Sequence ID: gb|AAF34793.1 andgb|AAA60394.1), which are shown respectively as SEQ ID NO 1 and 2:

(SEQ ID NO 1) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR(SEQ ID NO 2) RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR

As used herein, the term ‘variant’ refers to a polypeptide sequencewhich is a naturally occurring polymorphic form of the basic sequence aswell as synthetic variants, in which one or more amino acids within thechain are inserted, removed or replaced. However, the variant produces abiological effect which is similar to that of the basic sequence. Forexample, the variant mentioned above will act in a manner similar tothat of the intracellular domain of human CD3 [zeta] chain. Amino acidsubstitutions may be regarded as “conservative” where an amino acid isreplaced with a different amino acid in the same class with broadlysimilar properties. Non-conservative substitutions are where amino acidsare replaced with amino acids of a different type or class.

Amino acid classes are defined as follows:

Class Amino acid examples Nonpolar: A, V, L, I, P, M, F, W Unchargedpolar: G, S, T, C, Y, N, Q Acidic: D, E Basic: K, R, H.

As is well known to those skilled in the art, altering the primarystructure of a peptide by a conservative substitution may notsignificantly alter the activity of that peptide because the side-chainof the amino acid which is inserted into the sequence may be able toform similar bonds and contacts as the side chain of the amino acidwhich has been substituted out. This is so even when the substitution isin a region which is critical in determining the peptide's conformation.

Non-conservative substitutions may also be possible provided that thesedo not interrupt the function of the polypeptide as described above.Broadly speaking, fewer non-conservative substitutions will be possiblewithout altering the biological activity of the polypeptides.

In general, variants will have amino acid sequences that will be atleast 70%, for instance at least 71%, 75%, 79%, 81%, 84%, 87%, 90%, 93%,95%, 96% or 98% identical to the basic sequence, for example SEQ ID NO 1or SEQ ID NO 2, Identity in this context may be determined using theBLASTP computer program with SEQ ID NO 2 or a fragment, in particular afragment as described below, as the base sequence. The BLAST software ispublicly available.

The co-stimulatory signal sequence (b) is suitably located between thetransmembrane domain (c) and the signalling region (a) and remote fromthe binding element (d). Similarly the co-stimulatory signal sequence(e) is suitably located adjacent the transmembrane domain (f) and remotefrom the binding element (g).

Suitable co-stimulatory signalling regions for use as elements (b) and(e) above are also well known in the art, and include members of theB7/CD28 family such as B7-1, B7-2, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6,B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, PD-1, PD-L2 or PDCD6; or ILT/CD85family proteins such as LILRA3, LILRA4, LILRB1, LILRB2, LILRB3 orLILRB4; or tumour necrosis factor (TNF) superfamily members such as4-11BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT,Lymphotoxin-alpha, OX40, RELT, TACI, TL1A, TNF-alpha or TNF RII; ormembers of the SLAM family such as 2B4, BLAME, CD2, CD2F-10, CD48, CD8,CD84, CD229, CRACC, NTB-A or SLAM; or members of the TIM family such asTIM-1, TIM-3 or TIM-4; or other co-stimulatory molecules such as CD7,CD96, CD160, CD200, CD300a, CRTAM, DAP12, Dectin-1, DPPIV, EphB6,Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1. LAG-3 or TSLPR.

The selection of the co-stimulatory signalling regions may be selecteddepending upon the particular use intended for the transformed cells. Inparticular, the co-stimulatory signalling regions selected for (b) and(e) above are those which may work co-operatively or synergisticallytogether. For example, the co-stimulatory signalling regions for (b) and(e) may be selected from CD28, CD27, ICOS, 4-BB, OX40, CD30, GITR, HVEM,DR3 or CD40.

In a particular embodiment, one of (b) or (e) is CD28 and the other is4-18B or OX40.

In a particular embodiment, (b) is CD28.

In another particular embodiment (e) is 4-1BB or OX40 and in particular,is 4-1BB. In another embodiment, (e) is CD27.

The transmembrane domains of (c) and (f) above may be the same ordifferent but in particular are different to ensure separation of theconstructs on the surface of the cell. Selection of differenttransmembrane domains may also enhance stability of the vector sinceinclusion of a direct repeat nucleic acid sequence in the viral vectorrenders it prone to rearrangement, with deletion of sequences betweenthe direct repeats. Where the transmembrane domains of (c) and (f) arethe same however, this risk can be reduced by modifying or “wobbling”the codons selected to encode the same protein sequence.

Suitable transmembrane domains are known in the art and include forexample, CD8a, CD28, CD4 or CD3z transmembrane domains.

Where the co-stimulatory signalling region comprises CD28 as describedabove, the CD28 transmembrane domain represents a suitable option. Thefull length CD28 protein is a 220 amino acid protein of SEQ ID NO 3.

(SEQ ID NO 3) MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPG PTRKHYQPYAPPRDFAAYRSwhere the transmembrane domain is shown in bold type.

In particular, one of the co-stimulatory signalling regions is basedupon the hinge region and suitably also the transmembrane domain andendodomain of CD28. In particular, which comprises amino acids 114-220of SEQ ID NO 3, shown below as SEQ ID NO 4.

(SEQ ID NO 4) IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRS

In a particular embodiment, one of the co-stimulatory signalling regions(b) or (e) above is a modified form of SEQ ID NO 4 which includes ac-myc tag of SEQ ID No 5.

The c-myc tag is well known and is of SEQ ID NO 5.

(SEQ ID NO 5) EQKLISEEDL

The c-myc tag may be added to the co-stimulatory signalling region (b)or (e) by insertion into the ectodomain or by replacement of a region inthe ectodomain, which is therefore within the region of amino acids1-152 of SEQ ID NO 3.

In a particularly preferred embodiment, the c-myc tag replaces MYPPPYmotif in the CD28 sequence. This motif represents a potentiallyhazardous sequence. It is responsible for interactions between CD28 andits natural ligands, CD80 and CD86, so that it provides potential foroff-target toxicity when CAR T-cells encounter a target cell thatexpresses either of these ligands. By replacement of this motif with atag sequence as described above, the potential for unwanted side-effectsis reduced.

Thus in a particular embodiment, the co-stimulatory signalling region(b) of the construct is of SEQ ID NO 6.

(SEQ ID NO 6) IEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY APPRDFAAYRS

Furthermore, the inclusion of a c-myc epitope means that detection ofthe CAR T-cells using a monoclonal antibody is facilitated. This is veryuseful since flow cytometric detection had proven unreliable when usingsome available antibodies.

In addition, the provision of a c-myc epitope tag could facilitate theantigen independent expansion of targeted CAR T-cells, for example bycross-linking of the CAR using the appropriate monoclonal antibody,either in solution or immobilised onto a solid phase (e.g. a bag).

Moreover, expression of the epitope for the anti-human c-myc antibody,9e10, within the variable region of a TCR has previously been shown tobe sufficient to enable antibody-mediated and complement mediatedcytotoxicity both in vitro and in vivo (Kieback et al. (2008) Proc.Natl. Acad. Sci. USA, 105(2) 623-8). Thus, the provision of such epitopetags could also be used as a “suicide system”, whereby an antibody couldbe used to deplete CAR T-cells in vivo, in the event of toxicity.

The binding elements (d) and (g) will be different and will bind thesame, overlapping or different epitopes. In a particular embodiment thefirst and second epitopes are associated with the same receptor orantigen. Thus the first and second epitopes as described above may, insome cases, be the same, or overlapping so that the binding elements (d)and (g) will compete in their binding. Alternatively, the first andsecond epitopes may be different and associated with the same ordifferent antigens depending upon the particular therapy beingenvisaged. In one embodiment, the antigens are different but may beassociated with the same disease such as the same specific cancer.

As used herein, the term ‘antigen’ refers to any member of a specificbinding pair that will bind to the binding elements. Thus the termincludes receptors on target cells.

Thus suitable binding elements (d) and (g) may be any element whichprovides the pCAR with the ability to recognize a target of interest.The target to which the pCARs of the invention are directed can be anytarget of clinical interest to which it would be desirable to induce a Tcell response. This would include markers associated with cancers ofvarious types, including for example, one or more ErbB receptors or theα_(v)β₆ integrin, markers associated with prostate cancer (for exampleusing a binding element that binds to prostate-specific membrane antigen(PSMA)), breast cancer (for example using a binding element that targetsHer-2 (also known as ErbB2)) and neuroblastomas (for example using abinding element that targets GD2), melanomas, small cell or non-smallcell lung carcinoma, sarcomas and brain tumours. In a particularembodiment, the target is one or more ErbB dimers as described above orthe receptor for colony stimulating factor-1 (CSF-1R) or the α_(v)β₆integrin, all of which have been implicated in several solid tumours.

The binding elements used in the pCARs of the invention may compriseantibodies that recognize a selected target. For convenience, theantibody used as the binding element is preferably a single chainantibody (scFv) or single domain antibody, from a camelid, human orother species. Single chain antibodies may be cloned from the V regiongenes of a hybridoma specific for a desired target. The production ofsuch hybridomas has become routine, and the procedure will not berepeated here. A technique which can be used for cloning the variableregion heavy chain (VH) and variable region light chain (VL) has beendescribed in Orlandi et al., Proc. Nati Acad. Sci. (USA) 86: 3833-3837(1989). Briefly, mRNA is isolated from the hybridoma cell line, andreverse transcribed into complementary DNA (cDNA), for example using areverse transcriptase polymerase chain reaction (RT-PCR) kit.Sequence-specific primers corresponding to the sequence of the VH and VLgenes are used. Sequence analysis of the cloned products and comparisonto the known sequence for the VH and VL genes can be used to show thatthe cloned VH gene matched expectations. The VH and VL genes are thenattached together, for example using an oligonucleotide encoding a(gly4-ser)3 linker.

Alternatively, a binding element of a pCAR may comprise ligands such asthe T1E peptide (binds ErbB homo- and heterodimers), colony-stimulatingfactor-1 (CSF-1) or IL-34 (both bind to the CSF-1 receptor). The T1Epeptide is a chimeric fusion protein composed of the entire mature humanEGF protein, excluding the five most N-terminal amino acids (amino acids971-975 of pro-epidermal growth factor precursor (NP_001954.2)), whichhave been replaced by the seven most N-terminal amino acids of themature human TGF-α protein (amino acids 40-46 of pro-transforming growthfactor alpha isoform 1 (NP_003227.1)).

In another embodiment, a binding element of a pCAR comprises an α_(v)β₆integrin-specific binding agent. The integrin α_(v)β₆ is now regarded asa target in cancer as it has been found to be strongly upregulated inmany types of cancer. α_(v)β₆ has been identified as a receptor forfoot-and-mouth disease virus (FMDV) in vitro by binding through an RGDmotif in the viral capsid protein, VP1. As a result, as described forexample in U.S. Pat. No. 8,383,593, a range of peptides derived fromFMDV and in particular, peptides originating from the VP1 protein ofFMDV and comprising an RGD motif showed increased binding potency andbinding specificity. In particular, these peptides comprise the sequencemotif

(SEQ ID NO 7) RGDLX⁵X⁶L or (SEQ ID NO 8) RGDLX⁵X⁶I,wherein LX⁵X⁶L or LX⁵X⁶I is contained within an alpha helical structure,wherein X⁵ and X⁶ are helix promoting residues, which have aconformational preference greater than 1.0 for being found in the middleof an [alpha]-helix (from Creighton, 1993 and Pace C. N. and Scholtz J.M. (1998), Biophysical Journal, Vol. 75, pages 422-427). In particularsuch residues are independently selected from the group consisting ofGlu, Ala, Leu, Met, Gin, Lys, Arg, Val, lie, Trp, Phe and Asp.

Specific examples of such sequences include SEQ ID Nos 9-11 or variantsthereof:

(SEQ ID NO 9) YTASARGDLAHLTTTHARHL (SEQ ID NO 10) GFTTGRRGDLATIHGMNRPFor (SEQ ID NO 11) NAVPNLRGDLQVLAQKVARTThese peptides may form a particular group of binding elements for theCARs of the present application.

For selected malignancies such as Hodgkin's lymphoma and some breastcancers, two natural ligands are CSF-1 and IL-34 and these formparticularly suitable binding elements for (d) and (g). They do howeverbind with different affinities. The affinity of binding can impact onthe activity observed. It may be beneficial in this case to ensure thatthe binding element with the lower binding affinity is used as bindingelement (d) and that with the higher binding affinity is used as bindingelement (g). In particular, in an embodiment, the relative affinity ofthe second generation CAR (i) for its cognate target is lower than thatof the partnering TNFR-based chimeric co-stimulatory receptor (ii). Thisdoes not preclude the use of high or low affinity targeting moieties ineach position provided that this relative affinity relationship ismaintained. Thus in the case of the present invention, in a particularembodiment, binding element (d) is CSF-1 which has a relatively lowbinding affinity, whilst binding element (g) comprises IL-34 which has ahigher binding affinity.

Suitably the binding element is associated with a leader sequence whichfacilitates expression on the cell surface. Many leader sequences areknown in the art, and these include the macrophage colony stimulatingfactor receptor (FMS) leader sequence or CD124 leader sequence.

In a further embodiment, the cells expressing the pCAR are engineered toco-express a chimeric cytokine receptor, in particular the 4αβ chimericcytokine receptor. In 4αβ, the ectodomain of the IL-4 receptor-α chainis joined to the transmembrane and endodomains of IL-2/15 receptor-β.This allows the selective expansion and enrichment of the geneticallyengineered T-cells ex vivo by the culture of these cells in a suitablesupport medium, which, in the case of 4αβ, would comprise IL-4 as thesole cytokine support. Similarly, the system can be used with a chimericcytokine receptor in which the ectodomain of the IL-4 receptor-α chainis joined to the transmembrane and endodomains of another receptor thatis naturally bound by a cytokine that also binds to the common γ chain.

As discussed, these cells are useful in therapy to stimulate a T-cellmediated immune response to a target cell population. Thus a secondaspect of the invention provides a method for stimulating a T cellmediated immune response to a target cell population in a patient inneed thereof, said method comprising administering to the patient apopulation of immuno-responsive cells as described above, wherein thebinding elements (d) and (g) are specific for the target cell.

In a third aspect of the invention, there is provided a method forpreparing an immuno-responsive cell according to any one of thepreceding claims, said method comprising transducing a cell with a firstnucleic acid encoding a CAR of structure (i) as defined above; and alsoa second nucleic acid encoding a CAR of structure (ii) as defined above.

In particular, in this method, lymphocytes from a patient are transducedwith the nucleic acids encoding the CARs of (i) and (ii). In particular,T-cells are subjected to genetic modification, for example by retroviralmediated transduction, to introduce CAR coding nucleic acids into thehost T-cell genome, thereby permitting stable CAR expression. They maythen be reintroduced into the patient, optionally after expansion, toprovide a beneficial therapeutic effect. Where the cells such as theT-cells are engineered to co-express a chimeric cytokine receptor suchas 4αβ, the expansion step may include an ex vivo culture step in amedium which comprises the cytokine, such as a medium comprising IL-4 asthe sole cytokine support in the case of 4αβ. Alternatively, thechimeric cytokine receptor may comprise the ectodomain of the IL-4receptor-α chain joined to the endodomain used by a common γ cytokinewith distinct properties, such as IL-7. In this setting, expansion ofthe cells in IL-4 may result in less cell differentiation, capitalizingon the natural ability of IL-7 to achieve this effect. In this way,selective expansion and enrichment of genetically engineered T-cellswith the desired state of differentiation can be ensured.

In a fourth aspect of the invention, there is provided a combination ofa first nucleic acid encoding a CAR of (i) above and a second nucleicacid encoding a CCR of (ii) above. As indicated previously, thiscombination is referred to as a pCAR. Suitable sequences for the nucleicacids will be apparent to a skilled as person. The sequences may beoptimized for use in the required immuno-responsive cell. However, insome cases, as discussed above, codons may be varied from the optimum or‘wobbled’ in order to avoid repeat sequences. Particular examples ofsuch nucleic acids will encode the preferred embodiments describedabove.

In order to achieve transduction, the nucleic acids of the fourth aspectof the invention are suitably introduced into a vector, such as aplasmid or a retroviral vector. Such vectors including plasmid vectors,or cell lines containing them form a further aspect of the invention.

The first and second nucleic acids or vectors containing them may becombined in a kit, which is supplied with a view to generatingimmuno-responsive cells of the first aspect of the invention in situ.

Parallel chimeric activating receptors (pCAR) encoded by the nucleicacids described above form a further aspect of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be particularly described by way of example andwith reference to the following Figures in which:

FIG. 1A,B is a schematic diagram showing a panel of CARs and pCARs(named C34B and 34CB) embodying the invention. All CARs and pCARs wereco-expressed in the SFG retroviral vector with 4αβ, a chimeric cytokinereceptor in which the IL-4 receptor-α ectodomain has been fused to thetransmembrane and endodomain of IL-2 receptor-β. Use of 4αβ allowsselective enrichment and expansion of gene-modified T-cells by culturein IL-4, since it recruits the gamma c (γc) chain.

FIG. 2A,B shows the results of an experiment using CARs shown in FIG.1A,B. T-cells (1×10⁶ cells) expressing these CARs and pCARs (oruntransduced (UT) as control) were co-cultivated in vitro for 24 hourswith T47D tumour cells that express (T47D-FMS) or lack (T47D) thecognate target antigen (Colony-stimulating factor-1 receptor (CSF-1R),encoded by c-fms). Residual viable tumour cells were then quantified byMTT assay.

FIG. 3 shows a representative experiment in which T-cells that expressCARs and pCARs of FIG. 1A,B (or untransduced) T-cells as control) weresubjected to successive rounds of Ag stimulation in the absence ofexogenous cytokine. Stimulation was provided by weekly culture on T47DFMS monolayers and T-cell numbers were enumerated at the indicatedintervals.

FIG. 4 shows pooled data from 7 similar replicate experiments to thatshown in FIG. 3, indicating the fold expansion of CAR T-cells thatoccurred in the week after each cycle of stimulation.

FIG. 5 shows illustrative cytotoxicity assays performed at the time ofstimulation cycles 2, 6, 9 and 12 in the experiment shown in FIG. 3.This follows from the testing of T-cells for their ability to kill T47DFMS and unmodified T47D monolayers (MTT assay), twenty four hours afterthe time of each re-stimulation cycle.

FIG. 6 shows the results of testing of supernatant, removed fromcultures one day after each cycle of stimulation, for IL-2 and IFN-γcontent by ELISA.

FIG. 7A-D demonstrates the establishment of an in vivo xenograft modelof CSF-1R-expressing anaplastic large cell lymphoma, which allowedsubsequent testing of anti-tumour activity of CAR and pCAR-engineeredT-cells. The model was established using K299 cells, engineered toexpress firefly luciferase (luc) and red fluorescent protein (RFP). FIG.7A shows tumour formation following the intravenous injection of theindicated doses of K299 luc cells, quantified by bioluminescence imaging(BLI). Representative BLI images are shown in FIG. 7B in mice thatreceived 2 million tumour cells. Expression of RFP⁺ tumour cells (FIG.7C) in the indicated tissues are shown, demonstrating that tumours onlyformed in lymph nodes in this model. Expression of the CSF-1R on fiverepresentative lymph node tumours is shown in FIG. 7D.

FIG. 8A,B shows the results of therapeutic studies in which K299 luccells were injected intravenously in SCID Beige mice (n=9 per group,divided over 2 separate experiments). After 5 days, mice were treatedwith CAR T-cells. Pooled bioluminescence emission from tumours is shownin FIG. 8A. Bioluminescence emission from individual mice is shown inFIG. 8B and survival of mice shown in FIG. 8A.

FIG. 9 shows the weights of animals used in the therapeutic study overtime.

FIGS. 10-13 show the results of analysis of the expression of‘exhaustion markers’ from dual CAR (C34B) expressing T-cells of theinvention where FIG. shows the results for PD1 analysis, FIG. 11 showsthe results for TIM3 analysis, FIG. 12 shows the results of LAG3analysis and FIG. 13 shows the results for 2B4 analysis.

FIG. 14 is a schematic diagram of a panel of CARs and constructstargeted to the integrin αvβ6 which have been prepared including a pCAR(named SFG TIE-41BB/A20-28z) embodying the invention. A20-28z is asecond generation CAR that is targeted using the A20 peptide derivedfrom foot and mouth disease virus. A20 binds with high affinity to αvβ6and with 85-1000 fold lower affinity to other RGD-binding integrins.C20-28z is a matched control in which key elements of A20 have beenmutated to abrogate integrin binding activity. All CARs have beenco-expressed with 4αβ as described in FIG. 1A,B.

FIG. 15A,B is a series of histograms obtained by flow cytometryillustrating integrin expression in A375 puro and Panc1 cells. Cellswere stained with anti-β6 (Biogen Idec) followed by secondary anti-mousePE, anti-αvβ3 or anti-αvβ5 (both APC conjugated, Bio-Techne). Gates wereset based on secondary antibody alone or isotype controls.

FIG. 16A,B is a series of graphs illustrating the cytotoxicity of CARsincluding the pCARs of the invention targeted to αvβ6. T-cellsexpressing the indicated CARs and pCARs were co-cultivated withαvβ6-negative (Panc1 and A375 puro) or αvβ6-positive (Bxpc3 and A375puro β6) tumour cells. Data show the mean±SEM of 2-7 independentexperiments, each performed in triplicate. *p<0.05; **p<0.01;***p<0.001.

FIG. 17A,B is a series of graphs showing production of IFN-γ by CARsincluding pCARs of the invention, targeted to αvβ6. T-cells expressingthe indicated CARs and pCARs were co-cultivated with αvβ6-negative(Panc1 and A375 puro) or αvβ6-positive (Bxpc3 and A375 puro 016) tumourcells. Data show the mean±SEM of 5-6 independent experiments, eachperformed in duplicate. *p<0.05; **p<0.01; ***p<0.001; ns—notsignificant.

FIG. 18A,B shows the results of re-stimulation experiments using the CARand pCAR-engineered T-cells described above and indicating the abilityof A20-28z/T1E-41BB pCAR T-cells to undergo repeated antigenstimulation, accompanied by expansion of T-cells and destruction oftarget cells that do (Bxpc3) or do not (Panc1) express the αvβ6integrin.

FIG. 19A,B shows the results of re-stimulation experiments usingpCAR-engineered T-cells in which A20-28z was co-expressed with T1E-41BB,T1E-CD27 or T1E-CD40, allowing the comparative evaluation ofco-stimulation by additional members of the TNF receptor family. ControlT-cells were non-transduced (NT) while CARs contained truncated (tr)endodomains. T-cells were re-stimulated on target cells that do (Bxpc3)or do not (Panc1) express the αvβ6 integrin, making comparison withunstimulated T-cells. In the case of Bxpc3 cells, superior expansion(FIG. 19A) accompanied by sustained cytotoxic activity (FIG. 19B) wasobserved with A20-28z/T1E-CD27 T-cells. By contrast, with Panc1 cells,superior expansion (FIG. 19A) accompanied by sustained cytotoxicactivity (FIG. 19B) was observed with A20-28z/T1E-CD27 T-cells. Thesedata demonstrate that additional members of the TNF receptor family canalso deliver co-stimulation using the pCAR format.

EXAMPLE 1

A panel of CARs targeted against the CSF-1 receptor (encoded by c-FMS),which is over-expressed in Hodgkin's lymphoma, anaplastic large celllymphoma and some solid tumours such as triple negative breast cancerwere prepared and are illustrated schematically in FIG. 1A,B. The panelof CARs included both second and third generation CARs with either ofthe two natural ligands, CSF-1 or IL-34, as the targeting moieties.Although both CSF-1 and IL-34 bind to CSF-1 receptor, IL-34 binds withmuch higher affinity (34-fold higher than CSF-1).

The constructs SFG C28ζ and SFG CTr were cloned in the SFG retroviralvector as NcoI/XhoI fragments, ensuring that their start codons are atthe site of the naturally occurring NcoI site, previously occupied bythe deleted env gene. Gene expression is achieved from the Moloneymurine leukaemia virus (MoMLV) long terminal repeat (LTR), which haspromoter activity and virus packaging of the RNA is ensured by the MoMLVψ packaging signal, which is flanked by splice donor and acceptor sites.

All other constructs were designed and cloned using the PolymeraseIncomplete Primer Extension (PIPE) cloning method. PIPE cloning methodis a PCR-based alternative to conventional restriction enzyme- andligation-dependent cloning methods. It eliminates the need toincorporate restriction sites, which could encode additional unwantedresidues into expressed proteins. The PIPE method relies on theinefficiency of the amplification process in the final cycles of a PCRreaction, possibly due to the decreasing availability of dNTPs, whichresults in the generation of partially single-stranded (PIPE) PCRproducts with overhanging 5′ends. A set of vector-specific primers wasused for PCR vector linearization and another set of primers with5′-vector-end overlapping sequences then used for insert amplification,generating incomplete extension products by PIPE. In a following step,the PIPE products were mixed and the single-stranded overlappingsequences annealed and assembled as a complete SFG CAR construct.Successful cloning was confirmed by diagnostic restriction digestion.DNA sequencing was performed on all constructs to confirm that thepredicted coding sequence was present, without any PCR-induced mutations(Source Bioscience, UK).

The panel included two “dual targeted” Chimeric Activating Receptors(pCARS) in which CSF-1 or IL-34 are coupled to 28z and 4-1BB, or viceversa. The dual targeted pCAR combinations were then stoichiometricallyco-expressed in the same T-cell population using a Thosea Asigna(T)2A-containing retroviral vector. One of these CARs was designated‘C34B’ (CSF1-28z plus IL34-41BB) and the other was named ‘34CB’(IL34-28z plus CSF1-41BB).

In these dual targeted CAR T-cells, both co-stimulatory motifs(CD28/4-1BB) are placed in their natural location, close to themembrane, physically separated from each other and co-expressed in thesame T-cell.

All CARs were co-expressed with an IL-4 responsive 4αβ receptor using anadditional T2A element in the vector. This enables enrichment/expansionof T-cells using IL-4, making it easier to compare the function of thesediverse cell populations after selection.

The main focus of the experiments was to test the behaviour of theT-cells on repeated re-stimulation with tumour target cells that eitherexpress or lack the FMS/CSF-1 receptor target. In each cycle, 1 millionof the indicated IL-4 expanded CAR T-cells were suspended in RPMI+humanAB serum and cultured with a confluent monolayer (24 well dish) of theantigen-expressing target (T47D FMS) or antigen null target (T47D).

Thereafter, if the CAR T-cells had persisted and destroyed themonolayer, 1 million T-cells were removed and re-stimulated in anidentical manner each week. Total cell number was extrapolated at eachtime-point depending on the expansion of T-cells that occurred in eachweekly cycle.

Throughout all of these experiments, T-cells were cultured in theabsence of any exogenous cytokine such as IL-2 or IL-4—so they had tomake their own cytokines in order to persist and expand. Cytokine (IFN-γand IL-2) production was measured by ELISA in supernatants harvestedfrom T-cell/tumour cell co-cultures, providing a second marker ofeffective co-stimulation.

It was found (FIG. 2A,B) that on their first exposure to a tumourmonolayer that expresses target (FMS encoded CSF-1 receptor), all CARsthat are predicted to kill do so (pooled data from 12 expts). Thecontrols are UT (untransduced), P4 (targets an irrelevant antigen, PSMA)and CT4 in which the endodomain is truncated. As expected, none of theCAR T-cells kill tumour cells that lack CSF-1 receptor (T47D).

A representative re-stimulation experiment is shown in FIG. 3. Pooledre-stimulation data from 7 experiments is shown in FIG. 4. In this case,proliferation on the first cycle was similar for most of the constructs,although the IL-34 targeted second and third generation constructs werepoorer. This may be because the affinity of the IL-34 targeting moietyis too high.

In the later cycles however, the C34B dual pCAR combination (a CSF-1targeted 28z second generation CAR co-expressed with an IL-34 targeted4-1BB co-stimulatory motif) consistently emerged as clearly superior.

In the experiment shown in FIG. 3, supernatant was collected 24 hoursafter the time of each re-stimulation cycle and was analysed forcytokine content (IFN-γ and IL-2) by ELISA. The percentage of residualtumour cell viability was measured by MTT assay (representative examplesshown in FIG. 5). The cytokine production results are shown in FIG. 6.It was found that only the C34B CAR T-cells retained the ability to makeIL-2 throughout each cycle of stimulation. This was lost by all of theother CAR combinations after the first cycle. Sustained retention of theability to make IL-2 through recursive re-stimulation is not usuallyseen with CAR T-cells and this suggests that this delivery of dualco-stimulation is fundamentally altering the differentiation of thesecells in vitro, delaying the onset of anergy.

Number of viable T-cells post monolayer destruction on consecutivecycles of Ag-stimulation was also monitored and the results are shown inFIG. 5. After the second cycle of re-stimulation, all CARs except C34Bbegin to lose the ability to achieve CSF-1R-dependent tumour cellkilling. By contrast, T-cells that express C34B retain antigen-dependentpotency in this cytotoxic assay for up to 13 iterative cycles ofre-stimulation, but never elicit cytotoxicity against unmodified T47Dcells.

Also, so-called “exhaustion markers” on these T-cells (PD1, TIM3, 284and LAG3) were also measured by flow cytometry. The results are shown inFIGS. 10-13. As expected, the percentage of T cells that expressedvarious exhaustion markers progressively increased on the re-stimulatedT-cells, but this did not retard the proliferation, tumour celldestruction or cytokine release by the C34B cells, upon antigenstimulation. This suggests that the superior function of C34B is not theresult of delayed upregulation of exhaustion markers.

In summary, the pCAR approach of the invention seems to maintain thecells in a state whereby they retain responsiveness to antigen throughmore cycles of re-stimulation. There are indications that it may retarddifferentiation beyond controlled memory state and it appears to delaythe onset of anergy while retaining the ability of the cells to makeIL-2 upon activation.

EXAMPLE 2

Analysis of Effects In Vivo

A panel of CARs used in Example 1 above were tested for anti-tumouractivity using a highly aggressive in vivo xenograft model in which theCSF-1 receptor target is expressed at low levels and in which disease isdisseminated throughout lymph nodes (FIG. 7A-D). Tumour cells weretagged with firefly luciferase, allowing the non-invasive monitoring ofdisease burden.

SCID/Beige mice were randomised into 6 groups (9 animals per groupcombined over two independent experiments) and were inoculatedintravenously (IV) with 2×10⁶ K299 tumour cells, re-suspended in 200 μLPBS. On day 5, the groups were treated with one of the therapeuticregimens indicated below:

-   -   C4B group: 20×10⁶ C4B T-cells IV    -   C34B group: 20×10⁶ C34B T-cells IV    -   43428Bz: 20×10⁶ 43428Bz T-cells IV    -   34CB group: 20×10⁶ 34CB T-cells IV    -   UT (Untransduced) group: 20×10⁶ untransduced T-cells IV    -   NT (Non-treated) group: 200 μL PBS IV

Tumour growth was monitored using bioluminescence imaging (BLI) atappropriate time-points for the duration of the study.

The results are shown in FIG. 8A,B. Again, the best performing systemwas that of the pCAR, C34B, indicated by lower average BLI emission(FIG. 8A-B), delayed tumour progression or tumour regression, leading toprolonged survival of mice (FIG. 8A).

Animals were weighed throughout the experiment and no significanttoxicity was noted (FIG. 9).

EXAMPLE 3

Selection of targeting moieties to engineer pCARs that elicit T-cellactivation in an αvβ6-dependent manner.

A panel of CARs that target αvβ6 integrin alone or together with theextended ErbB family were prepared and are shown schematically in FIG.14. The binding element used in this case was A20 peptide (SEQ ID NO 11)derived from the GH-loop of the capsid protein VP1 from Foot and MouthDisease Virus (serotype 01 BFS)(U.S. Pat. No. 8,927,501). This wasplaced downstream of a CD124 signal peptide and fused to CD28 and CD3ζendodomains to form A20-28ζ, a 2nd generation CAR. A control (C20-28ζ)was prepared comprising a similar construct but with a scrambledtargeting peptide (named C20) in which the key RGDL motif was replacedwith AAAA. A second control comprised A20 fused to a CD28 truncatedendodomain (A20-Tr).

To create the pCAR of the invention (named TIE-41BB/A20-28z), A20-28zwas co-expressed with a chimeric co-stimulatory receptor comprising apan-ErbB targeted peptide (T1E) fused to a CD8a transmembrane and a 41BBendodomain.

Where indicated, CARs were co-expressed with the 4αβ chimeric cytokinereceptor to allow for IL-4-mediated enrichment in vitro. Equimolarco-expression of the IL-4-responsive 4αβ chimeric cytokine receptor, inwhich the IL-4 receptor α ectodomain is fused to the transmembrane andendodomain of the shared IL-2/15 receptor β, was achieved using a ThoseaAsigna (T)2A ribosomal skip peptide. These chimeric molecules wereexpressed in human T-cells by retroviral gene transfer.

The integrin expression pattern of cancer cell lines A375 was assessedusing flow cytometry (FIG. 15A,B), and these were separated intoαvβ6-negative (Panc1 and A375 puro) or αvβ6-positive (Bxpc3 and A375puro β6) tumour cells. These cells were co-cultured with CAR T-cells atan effector:target ratio of 1:1 for either 24, 28 or 72 hours, afterwhich time, cytotoxicity was assessed by MTT assay and expressedrelative to untreated tumour cells. The results are shown in FIG. 16A,B.

These data show that A20-28z CAR T-cells kill all target cells thatexpress αvβ6 integrin (Bxpc3 and A375 β6 puro), but spare targets thatlack this integrin (Panc1 and A375 puro). Secondly, the control CARsC20-28z and A20-Tr are inactive in these assays. Thirdly, T-cells thatexpress the T1E-41BB/A20-28z pCAR cause efficient killing of targetcells that express αvβ6 integrin (Bxpc3 and A375 β6 puro). All of theseresults are as expected. Notably however, T-cells that express theT1E-41BB/A20-28z pCAR also cause the killing of target cells that lackαvβ6 (Panc1 and A375 puro). This indicates that, within a pCARconfiguration, the ability of the A20 peptide to bind non-αvβ6 integrinswith low affinity is sufficient to trigger the activation of theseengineered T-cells.

Production of IFN-γ by the pCAR and control engineered T-cells was thenassessed. Tumour cells that lacked αvβ6 (Panc1 and A375 puro) orexpressed αvβ6 (Bxpc3 and A375 puro 36) were co-cultured withgenetically engineered T-cells at an effector:target ratio of 1:1 andsupernatant was collected after 24, 48 or 72 hours. Levels of IFN-γ werequantified by ELISA (eBioscience). The results are shown in FIG. 17A,B.As expected, the controls did not generate significant quantities ofIFN-γ while A20-28z CAR T-cells released IFN-γ when cultured withαvβ6-positive (Bxpc3 and A375 puro 136) tumour cells. Notably, T-cellsthat express the pCAR of the invention, TIE-41BB/A20z, produce moreIFN-γ than A20-28z T-cells when cultured with αvβ6-positive (Bxpc3)tumour cells. In addition, TIE-41BB/A20z⁺ T-cells produced IFN-γ whencultured with αvβ6-negative (Panc1 and A375 puro) tumour cells. Onceagain, this demonstrates that, within a pCAR configuration, low affinitybinding of the A20 peptide to non-αvβ6 integrins is sufficient totrigger the activation of these engineered T-cells.

Next, the CAR T-cell populations were re-stimulated bi-weekly in theabsence of IL-2 support on Panc1 (αvβ6 negative) or Bxpc3 tumour cells(αvβ6 positive). Tumour cells were co-cultured with CAR T-cells derivedfrom a patient with pancreatic ductal adenocarcinoma (PDAC) at aneffector:target ratio of 1:1 (FIG. 18A,B). T-cells were initially addedat 2×10⁵ cells/well and were counted 72 hrs after co-culture to assessexpansion (top panels). Cytotoxicity was assessed at 72 hrspost-addition of T-cells by MTT assay (bottom panels). If there were asufficient number of T-cells (2×10⁵), T-cells were re-stimulated on afresh tumour monolayer and the process repeated a further 72 hrs later.

Results are shown in FIG. 18A,B. These illustrate that A20-28z/T1E-41BB⁺T-cells undergo a number of rounds of expansion accompanied by IL-2release (data not shown) and destruction of αvβ6⁺ Bxpc3 cells. Onceagain, they also underwent a number of rounds of expansion accompaniedby IL-2 release and destruction of Panel tumour cells.

Overall, the results clearly showed that the pCAR comprisingA20-28z/T1E-41BB exhibits enhanced in vitro functionality compared to a2^(nd) generation CAR targeted against αvβ6. Furthermore, theA20-28z/T1E-41BB⁺ T-cells also undergo activation by Panc1 or A375 purocells, which express minimal to undetectable levels of this integrin.Taken with the findings obtained using the C34B pCAR (examples 1 and 2),this indicates that the pCAR configuration allows T-cell activation tooccur upon serial re-stimulation when a high affinity bindinginteraction occurs with the 41BB CCR while a lower affinity interactionoccurs with the 28z 2^(nd) generation CAR.

EXAMPLE 4

Use of an alternative TNF receptor family member, CD27 to engineer afunctional pCAR.

Using the A20-28z/T1E-41BB pCAR as starting material, additional pCARswere engineered in which the 41BB module was replaced by alternativemembers of the TNF receptor family, namely CD27 or CD40. Control pCARswere engineered in which endodomains were truncated (tr). Target cellsthat express (Bxpc3) or lack (Panc1) αvβ6 were plated at a density of5×10⁴ cells per well of a 24 well plate. After 24 hours, 5×10⁴ pCART-cells were added to target cells or empty wells (“unstimulated”),without exogenous cytokine support. After a further 72 hours, T-cellswere harvested from the wells and were counted (FIG. 19A). An MTT assaywas performed to determine the percentage viability of the residualtarget cells, making comparison with control target cells that had beenplated without addition of T-cells (FIG. 19B). If T-cells proliferatedafter each cycle of stimulation, they were re-stimulated on fresh targetcells, exactly as described above. Proliferation of pCAR T-cells (FIG.19A) and MTT assay (FIG. 19B) were performed after 72 hours as before.Iterative re-stimulation of pCAR T-cells and assessment of target cellkilling was continued in this manner until T-cells no longerproliferated over the course of each 72 hour cycle.

These data once again confirm the superior functionality of theA20-28z/T1E-41BB pCAR when T-cells are stimulated on Panc1 target cells,indicated by sustained T-cell proliferation and tumour cell killing.This provides further confirmation that low affinity binding of the A20peptide to non-αvβ6 integrins is sufficient to trigger the activation ofthese engineered T-cells. Notably however, the A20-28z/T1E-CD27 pCARachieved the greatest level of proliferation (FIG. 19A) and sustainedtumour cell killing (FIG. 19B) when re-stimulated on αvβ6-expressingBxpc3 cells. By contrast, CD40-based pCARs exhibited modest function inthese assays. Together, these data demonstrate that a number of TNFreceptor family members can be employed to engineer pCARs thatdemonstrate superior functionality, exemplified by CD27 or 41BB.

The invention claimed is:
 1. A combination of a first nucleic acidencoding a second generation chimeric antigen receptor (CAR) and asecond nucleic acid encoding a chimeric costimulatory receptor, wherein:(i) the second generation CAR comprises: (a) a signalling region; (b) aco-stimulatory signalling region; (c) a transmembrane domain; and (d) abinding element that specifically interacts with a first epitope on atarget antigen; and (ii) the chimeric costimulatory receptor comprises:(e) a co-stimulatory signalling region which is different to that of(b); (f) a transmembrane domain; and (g) a binding element thatspecifically interacts with a second epitope on a target antigen.
 2. Thecombination of claim 1, wherein the first and second nucleic acids areincluded within a single polynucleotide.
 3. The combination of claim 1,wherein the signalling region (a) comprises the intracellular domain ofhuman CD3ζ [zeta] chain or a variant thereof.
 4. The combination ofclaim 1, wherein the co-stimulatory signalling regions (b) and (e) arechosen from CD28, CD27, ICOS, 4-1BB, OX40, CD30, GITR, HVEM, DR3, andCD40.
 5. The combination of claim 4, wherein one of (b) or (e) is CD28and the other of (b) or (e) is 4-1BB or OX40.
 6. The combination ofclaim 5, wherein (b) is CD28.
 7. The combination of claim 5, wherein (e)is 4-1BB or CD28.
 8. The combination of claim 1, wherein thetransmembrane domains (c) and (f) independently are CD8α or CD28transmembrane domains.
 9. The combination of claim 1, wherein the firstand second epitopes are associated with the same antigen.
 10. Thecombination of claim 1, which further encodes a chimeric cytokinereceptor.
 11. The combination of claim 10, wherein the chimeric cytokinereceptor is 4αβ.
 12. The combination of claim 1, wherein at least one ofthe binding elements (d) or (g) is a ligand for an ErbB dimer, areceptor for colony stimulating factor-1 (CSF-1R), or an α_(v)β₆integrin-specific binding agent.
 13. The combination of claim 1, whereinthe binding element (d) comprises CSF-1 and the binding element (g)comprises IL-34.
 14. The combination of claim 1, wherein the bindingelement (d) is an α_(v)β₆ integrin-specific binding agent which is apeptide comprising the sequence motif (SEQ ID NO 7) RGDLX⁵X⁶L or(SEQ ID NO 8) RGDLX⁵X⁶I,

wherein LX⁵X⁶L or LX⁵X⁶I is contained within an alpha helical structure,X⁵ and X⁶ are helix promoting residues; and the binding element (g) is aT1E peptide.
 15. The combination of claim 1 wherein the binding element(b) has a binding affinity that is lower than that of the bindingelement (g).